A Noninvasive In Vitro Monitoring System Reporting Skeletal Muscle Differentiation.
Tissue Eng Part C Methods
; 23(1): 1-11, 2017 01.
Article
en En
| MEDLINE
| ID: mdl-27901409
Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Diferenciación Celular
/
Músculo Esquelético
/
Creatina Quinasa
/
Desarrollo de Músculos
/
Proteínas Fluorescentes Verdes
/
Microscopía Fluorescente
Límite:
Animals
Idioma:
En
Revista:
Tissue Eng Part C Methods
Asunto de la revista:
BIOTECNOLOGIA
/
HISTOLOGIA
Año:
2017
Tipo del documento:
Article
País de afiliación:
Austria
Pais de publicación:
Estados Unidos